Rationale: It has been over a decade since drugs addressing the growth/migration of scar-forming fibroblasts were launched for idiopathic pulmonary fibrosis (IPF) and beyond. We hypothesize that novel, differentiated therapies must address the complexity of IPF pathobiology by targeting multiple cell types and pathways. Recent positive phase III trial data for the preferential PDE4B inhibitor nerandomilast, and accompanying preclinical mechanism of action studies, provide the first definitive evidence that targets which impact cell-types in addition to the fibroblast can be efficacious in the context of IPF. Objectives: By further defining the multiple mechanisms through which nerandomilast may confer efficacy, can we build on this knowledge by applying novel analytics and state-of-the-art technologies to guide the next generation of pharmacologic interventions for IPF? Methods and Results: We used a wide array of patient cells stimulated with an IPF mediator cocktail to understand effects of nerandomilast on epithelial cell activation; myofibroblast contractility and (de)differentiation; endothelial barrier integrity and immune cell adhesion/infiltration. Nerandomilast significantly reduced the release of biomarkers of fibrogenesis from small airway epithelial cell cultures. Myofibroblast contractility was also inhibited, accompanied by markers of dedifferentiation toward a more normal fibroblast. Furthermore, nerandomilast significantly reduced microvascular permeability as well as innate immune cell adhesion and infiltration in an adeno-associated virus-human diphtheria toxin receptor/diphtheria toxin mouse model of acute lung injury. These effects were linked to activated cAMP-associated pathways, G-protein-coupled receptor (GPCR) signaling events, mitogen-activated protein kinase (MAPK) signaling pathways and transforming growth factor beta 1(TGFβ1) signaling. For next generation target finding, we assessed all the above cell populations and more, using spatial multi-omic analyses of human disease samples to characterize pathologic crosstalk between multiple cell-types in disease-associated niches. We then applied AI-assisted systems – a gene prioritization algorithm (GPA) and large language model – to provide prioritized targets aligned with a particular pathologic niche. Finally, we explore the advances in in vitro co-culture / organoid test systems which are designed to functionally assess, and thereby help validate these targets of the future. Conclusions: Nerandomilast's efficacy in IPF may be attributable to effects on an array of patho-mechanisms in epithelial, endothelial, and immune cells, in addition to fibroblasts. Cutting edge technologies can build on such principles to transform drug discovery strategies. Next generation targets will influence our newly characterized pathologic niches, with the bold aim to not only limit disease but return functionality to the lung.

Previous studies have shown that altered AXL signaling is implicated in various diseases, with GAS6 recognized as its only relevant ligand to date. In this study, we show for the first-time a direct interaction between AXL and PROS1 using biochemical methods. Furthermore, we validate the biological significance of PROS1-AXL interaction through advanced quantitative and functional spatial imaging in both murine lung tissue, as well as human lung samples of idiopathic pulmonary fibrosis (IPF) patients. Our findings reveal the role of AXL-mediated biology in alveolar repair and the fibrotic response driven by GAS6 as well as PROS1. Notably, this effect involves PROS1 interacting with AXL to counteract GAS6 mediated effects. Together with the distinct temporal expression of profibrotic genes and the interplay between AXL and TGF-ß pathway, this emphasizes the potential of targeting AXL mediated biology for therapeutic intervention in IPF to allow alveolar restoration. ### Competing Interest Statement The authors have declared no competing interest.

BACKGROUND:Idiopathic pulmonary fibrosis (IPF) is a progressive and debilitating respiratory disease with limited therapeutic options. Genetic association studies for IPF have identified several associations and probable effector genes that could not only help understanding IPF pathogenesis but also develop effective treatments. Assessing genetic overlap between IPF and severe COVID-19, an acute respiratory disease that can trigger pulmonary fibrosis, may reveal shared aetiology and mechanisms, thereby supporting the development of common treatments. METHODS:We carried out genome-wide association studies (GWAS), post-GWAS, and rare variant analyses using whole genome sequencing data from the 100,000 Genomes Project IPF cohort (n = 586). We performed a meta-analysis combining 100 kGP with published IPF GWASs (total 11,746 cases and 1,416,493 controls). We tested inhibition in vitro for a probable effector gene of an identified association. We also investigated genetic colocalisation between IPF and severe COVID-19 and leveraged their genetic correlation through multi-trait meta-analysis for discovery. FINDINGS:IPF meta-analysis identified an additional association at 1q21.2 (rs16837903, OR [95% CI] = 0.88 [0.85, 0.92], P = 9.5 × 10-9), which was replicated in independent data. MCL1, one of the probable effector genes of the 1q21.2 signal has a known antiapoptotic role, but MCL1 inhibition in vitro did not selectively deplete senescent alveolar epithelial cells. Rare variant burden analysis identified ANGPTL7, a secreted glycoprotein involved in the regulation of angiogenesis, as an IPF candidate gene (OR [95% CI] = 28.8 [8.51, 97.4], P = 6.7 × 10-8). We discovered additional shared genetic loci between IPF and severe COVID-19 at 1q21.2, 6p24.3, and 16p13.3, with probable effector genes MCL1, DSP, and RHBDF1, implicating regulation of apoptosis, cell adhesion, and epidermal growth factor signalling, respectively. The genetic correlation between IPF and severe COVID-19 was rg [95% CI] = 0.39 [0.25, 0.53]. Using multi-trait meta-analysis, we identified and replicated an additional candidate IPF signal at 2p16.1 with probable effector gene BCL11A, a regulator of haematopoiesis and lymphocyte development. INTERPRETATION:These findings prioritise probable effector genes mediating IPF risk and identify potential therapeutic targets that require validation, with genes colocalising with severe COVID-19 suggesting potential for developing common treatments. FUNDING:None.

Advancements in single-cell RNA sequencing (scRNA-seq) have revealed the phenotypic and functional diversity of tumor-associated macrophages (TAMs), identifying specific populations that directly impact the antitumor response. However, despite the recognition of TAMs as promising therapeutic targets for cancer treatment, research is hindered by the lack of validated human preclinical models. Here, we applied scRNA-seq to a 3D human cell-based model comprising tumor cell line-derived spheroids, cancer-associated fibroblasts and primary monocytes, a setup widely used in immuno-oncology research. Integration of our in vitro data with publicly available patient-derived datasets showed that the macrophages in this model share phenotypic characteristics with the pro-angiogenic and pro-fibrotic SPP1+ TAM population recently found across multiple cancer types and inflammatory lung diseases. This population was linked to aspects of disease progression and associated with poor prognosis in several tumor indications, highlighting the need for relevant models enabling its study as an immunotherapy target. Our research validates the use of a 3D human cell-based culture as a more in vivo-relevant model and enables the preclinical testing of novel macrophage-targeting drugs in a human disease-relevant setup.

Idiopathic pulmonary fibrosis (IPF) is a lethal disease with substantial unmet medical needs. While aberrant epithelial remodeling is a key factor in IPF progression, the molecular mechanisms behind this process remain elusive. Harnessing a 3D patient-derived organoid model and multi-omics approach, the first inventory of the connection between metabolic alteration, chromatin accessibility, and transcriptional regulation in IPF aberrant epithelial remodeling is provided. This remodeling is characterized by an increase in chromatin accessibility, particularly at JUNB motif-enriched promoter regions proximal to transcription start sites of metabolic and pro-fibrotic genes. Mechanistically, JUNB undergoes O-linked β-N-acetylglucosamine modification (O-GlcNAcylation), a critical step in modulating pro-fibrotic responses to chronic injury. This modification is pivotal in fostering the emergence of aberrant epithelial basal cells in the alveolar niche, a proposed driver of IPF pathology. Specific deletion of O-GlcNAcylation sites on JUNB attenuates the metaplastic differentiation of basal cells, thereby aiding in the restoration of the alveolar lineage. Together, the findings reveal a novel link between metabolic dysregulation and cell fate regulation at the chromatin level in fibrosis, mediated by the O-GlcNAc-JUNB axis, suggesting avenues for the development of new therapeutic strategies in IPF.

Despite advancements in antifibrotic therapy, idiopathic pulmonary fibrosis (IPF) remains a medical condition with unmet needs. Single-cell RNA sequencing (scRNA-seq) has enhanced our understanding of IPF but lacks the cellular tissue context and gene expression localization that spatial transcriptomics provides. To bridge this gap, we profiled IPF and control patient lung tissue using spatial transcriptomics, integrating the data with an IPF scRNA-seq atlas. We identified three disease-associated niches with unique cellular compositions and localizations. These include a fibrotic niche, consisting of myofibroblasts and aberrant basaloid cells, located around airways and adjacent to an airway macrophage niche in the lumen, containing SPP1 + macrophages. In addition, we identified an immune niche, characterized by distinct lymphoid cell foci in fibrotic tissue, surrounded by remodeled endothelial vessels. This spatial characterization of IPF niches will facilitate the identification of drug targets that disrupt disease-driving niches and aid in the development of disease relevant in vitro models.

Bleomycin-induced pulmonary fibrosis in mice mimics major hallmarks of idiopathic pulmonary fibrosis. Yet in this model, it spontaneously resolves over time. We studied molecular mechanisms of fibrosis resolution and lung repair, focusing on transcriptional and proteomic signatures and the effect of aging. Old mice showed incomplete and delayed lung function recovery 8 weeks after bleomycin instillation. This shift in structural and functional repair in old bleomycin-treated mice was reflected in a temporal shift in gene and protein expression. We reveal gene signatures and signaling pathways that underpin the lung repair process. Importantly, the downregulation of WNT, BMP, and TGFβ antagonists Frzb, Sfrp1, Dkk2, Grem1, Fst, Fstl1, and Inhba correlated with lung function improvement. Those genes constitute a network with functions in stem cell pathways, wound, and pulmonary healing. We suggest that insufficient and delayed downregulation of those antagonists during fibrosis resolution in old mice explains the impaired regenerative outcome. Together, we identified signaling pathway molecules with relevance to lung regeneration that should be tested in-depth experimentally as potential therapeutic targets for pulmonary fibrosis.

Profibrotic and prohomeostatic macrophage phenotypes remain ill-defined, both in vivo and in vitro, impeding the successful development of drugs that reprogram macrophages as an attractive therapeutic approach to manage fibrotic disease. The goal of this study was to reveal profibrotic and prohomeostatic macrophage phenotypes that could guide the design of new therapeutic approaches targeting macrophages to treat fibrotic disease. This study used nintedanib, a broad kinase inhibitor approved for idiopathic pulmonary fibrosis, to dissect lung macrophage phenotypes during fibrosis-linked inflammation by combining in vivo and in vitro bulk and single-cell RNA-sequencing approaches. In the bleomycin model, nintedanib drove the expression of IL-4/IL-13-associated genes important for tissue regeneration and repair at early and late time points in lung macrophages. These findings were replicated in vitro in mouse primary bone marrow-derived macrophages exposed to IL-4/IL-13 and nintedanib. In addition, nintedanib promoted the expression of IL-4/IL-13 pathway genes in human macrophages in vitro. The molecular mechanism was connected to inhibition of the colony stimulating factor 1 (CSF1) receptor in both human and mouse macrophages. Moreover, nintedanib counterbalanced the effects of TNF on IL-4/IL-13 in macrophages to promote expression of IL-4/IL-13-regulated tissue repair genes in fibrotic contexts in vivo and in vitro. This study demonstrates that one of nintedanib's antifibrotic mechanisms is to increase IL-4 signaling in macrophages through inhibition of the CSF1 receptor, resulting in the promotion of tissue repair phenotypes.

In many solid cancers, tumor-associated macrophages (TAM) represent the predominant myeloid cell population. Antigen (Ag) cross-presentation leading to tumor Ag–directed cytotoxic CD8+ T cell responses is crucial for antitumor immunity. However, the role of recruited monocyte-derived macrophages, including TAM, as potential cross-presenting cells is not well understood. Here, we show that primary human as well as mouse CD206+ macrophages are effective in functional cross-presentation of soluble self-Ag and non–self-Ag, including tumor-associated Ag (TAA), as well as viral Ag. To confirm the presence of cross-presenting TAM in vivo, we performed phenotypic and functional analysis of TAM from B16-F10 and CT26 syngeneic tumor models and have identified CD11b+F4/80hiCD206+ TAM to effectively cross-present TAA. We show that CD11b+CD206+ TAM represent the dominant tumor-infiltrating myeloid cell population, expressing a unique cell surface repertoire, promoting Ag cross-presentation and Ag-specific CD8+ T cell activation comparable with cross-presenting CLEC9A+ DCs (cDC1). The presence of cross-presenting CD206+ TAM is associated with reduced tumor burden in mouse syngeneic tumor models and with improved overall survival in cutaneous melanoma patients. Therefore, the demonstration of effective Ag cross-presentation capabilities of CD206+ TAM, including their clinical relevance, expands our understanding of TAM phenotypic diversity and functional versatility.

Airway mucociliary regeneration and function are key players for airway defense and are impaired in chronic obstructive pulmonary disease (COPD). Using transcriptome analysis in COPD-derived bronchial biopsies, we observed a positive correlation between cilia-related genes and microRNA-449 (miR449). In vitro, miR449 was strongly increased during airway epithelial mucociliary differentiation. In vivo, miR449 was upregulated during recovery from chemical or infective insults. miR0449−/− mice (both alleles are deleted) showed impaired ciliated epithelial regeneration after naphthalene and Haemophilus influenzae exposure, accompanied by more intense inflammation and emphysematous manifestations of COPD. The latter occurred spontaneously in aged miR449−/− mice. We identified Aurora kinase A and its effector target HDAC6 as key mediators in miR449-regulated ciliary homeostasis and epithelial regeneration. Aurora kinase A is downregulated upon miR449 overexpression in vitro and upregulated in miR449−/− mouse lungs. Accordingly, imaging studies showed profoundly altered cilia length and morphology accompanied by reduced mucociliary clearance. Pharmacological inhibition of HDAC6 rescued cilia length and coverage in miR449−/− cells, consistent with its tubulin-deacetylating function. Altogether, our study establishes a link between miR449, ciliary dysfunction, and COPD pathogenesis.

Next-generation sequencing (NGS) has revolutionized genomics, decreasing sequencing costs and allowing researchers to draw correlations between diseases and DNA or RNA changes. Technical advances have enabled the analysis of RNA expression changes between single cells within a heterogeneous population, known as single-cell RNA-seq (scRNA-seq). Despite resolving transcriptomes of cellular subpopulations, scRNA-seq has not replaced RNA-seq, due to higher costs and longer hands-on time. Here, we developed an automated workflow to increase throughput (up to 48 reactions) and to reduce by 75% the hands-on time of scRNA-seq library preparation, using the 10X Genomics Single Cell 3’ kit. After gel bead-in-emulsion (GEM) generation on the 10X Genomics Chromium Controller, cDNA amplification was performed, and the product was normalized and subjected to either the manual, standard library preparation method or a fully automated, walk-away method using a Biomek i7 Hybrid liquid handler. Control metrics showed that both quantity and quality of the single-cell gene expression libraries generated were equivalent in size and yield. Key scRNA-seq downstream quality metrics, such as unique molecular identifiers count, mitochondrial RNA content, and cell and gene counts, further showed high correlations between automated and manual workflows. Using the UMAP dimensionality reduction technique to visualize all cells, we were able to further correlate the results observed between the manual and automated methods (R=0.971). The method developed here allows for the fast, error-free, and reproducible multiplex generation of high-quality single-cell gene expression libraries.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive fibrosing interstitial lung disease with poor prognosis. The current standard-of-care for IPF is the kinase inhibitor nintedanib, which has a distinct inhibitory profile targeting a range of tyrosine kinases. The mechanism of action of nintedanib has been well characterized for myofibroblast activity, yet remains less clear for the immune populations distributed throughout the lung. Given the proposed role of heterogeneous pulmonary macrophage populations in mediating both protective and pathogenic roles in lung fibrosis, we sought to identify repair-associated macrophage populations in mice influenced by nintedanib after 7 and 14 days post-bleomycin challenge using single cell RNA sequencing. Bleomycin exposure triggered expansion of inflammatory MHCIIhigh macrophage populations, which was partially reversed after nintedanib treatment. Concurrently, nintedanib promoted the expansion of MHCIIlow macrophages, which were linked to attenuation of lung fibrosis. Concomitantly, nintedanib promoted an increased expression of canonical macrophage repair markers in MHCIIlow macrophages. Finally, exposure of inflammatory macrophages to nintedanib in vitro resulted in attenuated expression of MHCII transcripts. In conclusion, a component of nintedanib’s protective mode of action in lung fibrosis relies on expanding distinct MHCIIlow macrophage populations and redirecting them toward a reparative phenotype. This study provides a rationale for further refinement of therapeutic kinase inhibition in fibrotic diseases.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive fibrosing interstitial lung disease with poor prognosis. The current standard-of-care for IPF is the kinase inhibitor nintedanib, which has a distinct inhibitory profile targeting a range of tyrosine kinases. The mechanism of action of nintedanib has been well characterized for myofibroblast activity, yet remains less clear for the immune populations distributed throughout the lung. Given the proposed role of heterogeneous pulmonary macrophage populations in mediating both protective and pathogenic roles in lung fibrosis, we sought to identify repair-associated macrophage populations in mice influenced by nintedanib after 7 and 14 days post-bleomycin challenge using single cell RNA sequencing. Bleomycin exposure triggered expansion of inflammatory MHCIIhigh macrophage populations, which was partially reversed after nintedanib treatment. Concurrently, nintedanib promoted the expansion of MHCIIlow macrophages, which were linked to attenuation of lung fibrosis. Concomitantly, nintedanib promoted an increased expression of canonical macrophage repair markers in MHCIIlow macrophages. Finally, exposure of inflammatory macrophages to nintedanib in vitro resulted in attenuated expression of MHCII transcripts. In conclusion, a component of nintedanib’s protective mode of action in lung fibrosis relies on expanding distinct MHCIIlow macrophage populations and redirecting them toward a reparative phenotype. This study provides a rationale for further refinement of therapeutic kinase inhibition in fibrotic diseases.

Background: The vast ecosystem of single-cell RNA-sequencing tools has until recently been plagued by an excess of diverging analysis strategies, inconsistent file formats, and compatibility issues between different software suites. The uptake of 10x Genomics datasets has begun to calm this diversity, and the bioinformatics community leans once more towards the large computing requirements and the statistically driven methods needed to process and understand these ever-growing datasets. Results: Here we outline several Galaxy workflows and learning resources for single-cell RNA-sequencing, with the aim of providing a comprehensive analysis environment paired with a thorough user learning experience that bridges the knowledge gap between the computational methods and the underlying cell biology. The Galaxy reproducible bioinformatics framework provides tools, workflows, and trainings that not only enable users to perform 1-click 10x preprocessing but also empower them to demultiplex raw sequencing from custom tagged and full-length sequencing protocols. The downstream analysis supports a range of high-quality interoperable suites separated into common stages of analysis: inspection, filtering, normalization, confounder removal, and clustering. The teaching resources cover concepts from computer science to cell biology. Access to all resources is provided at the singlecell.usegalaxy.eu portal. Conclusions: The reproducible and training-oriented Galaxy framework provides a sustainable high-performance computing environment for users to run flexible analyses on both 10x and alternative platforms. The tutorials from the Galaxy Training Network along with the frequent training workshops hosted by the Galaxy community provide a means for users to learn, publish, and teach single-cell RNA-sequencing analysis.

Abstract Motivation Generating publication ready plots to display multiple genomic tracks can pose a serious challenge. Making desirable and accurate figures requires considerable effort. This is usually done by hand or using a vector graphic software. Results pyGenomeTracks (PGT) is a modular plotting tool that easily combines multiple tracks. It enables a reproducible and standardized generation of highly customizable and publication ready images. Availability and implementation PGT is available through a graphical interface on https://usegalaxy.eu and through the command line. It is provided on conda via the bioconda channel, on pip and it is openly developed on github: https://github.com/deeptools/pyGenomeTracks. Supplementary information Supplementary data are available at Bioinformatics online.

Vaccination has reduced morbidity and mortality of many diseases that previously caused devastating epidemics and deaths globally. Vaccines as a biological product may contain microorganisms or their derivatives. This aspect together with the fact that they are administered to healthy individuals (mainly children) means that approximately 70% of vaccines development time is dedicated to quality control. Monoclonal antibodies (MAbs) have become essential analytical tools for application in ELISAs, Western and Dot blotting, immunoprecipitation, and flow cytometric assays that ensure the quality control of vaccines. The aim of this work is to present a review of the methods used to obtain a platform of MAbs against Neisseria meningitidis polysaccharide antigens to use as an analytical tool for quality control of anti-meningococcal polysaccharide (Ps) vaccines. The MAbs obtained are used in five sandwich ELISAs developed for Ps quantification. The assays showed good reproducibility and repeatability, with quantitation and detection limits below 1 ng/mL. Dot Blot, as the Identity test of the Ps vaccine, was carried out to positively identify licensed and experimental vaccines. All assays described are suitable for the screening of multiple vaccine samples and could be useful for monitoring lot-to-lot consistency and stability.

Genome rearrangements that occur during evolution impose major challenges on regulatory mechanisms that rely on three-dimensional genome architecture. Here, we developed a scaffolding algorithm and generated chromosome-length assemblies from Hi-C data for studying genome topology in three distantly related Drosophila species. We observe extensive genome shuffling between these species with one synteny breakpoint after approximately every six genes. A/B compartments, a set of large gene-dense topologically associating domains (TADs), and spatial contacts between high-affinity sites (HAS) located on the X chromosome are maintained over 40 million years, indicating architectural conservation at various hierarchies. Evolutionary conserved genes cluster in the vicinity of HAS, while HAS locations appear evolutionarily flexible, thus uncoupling functional requirement of dosage compensation from individual positions on the linear X chromosome. Therefore, 3D architecture is preserved even in scenarios of thousands of rearrangements highlighting its relevance for essential processes such as dosage compensation of the X chromosome.